Comprehensive experimental information on the essential contributions of intramolecular dynamics to the biological function of proteins is critical for biophysical theories of equilibrium properties, such as heat capacity and thermal stability; for mechanistic interpretations of kinetic processes, such as enzyme catalysis, ligand recognition and protein folding; for the de novo synthesis of proteins; and for design of novel protein ligands, including pharmaceutical agents. The long- term goal of this research is to define the molecular determinants of stability and biological activity of ribonuclease HI (RNase H) by comparing the structural, dynamical and enzymatic properties of homologous proteins derived from escherichia coli and the extremely thermophilic bacterium Thermus thermophilus. RNase H is an endonuclease that hydrolyzes the RNA moiety in RNA-DNA hybrid oligonucleotides. The enzyme is distributed widely in prokaryotes and eukaryotes, and retroviral reverse transcriptase contains a C-terminal RNase H domain. RNase H is required for reverse transcription of retroviral RNA, may control the origin of replication in E. coli, has been implicated in the synthesis of multicopy single stranded DNA in gram negative bacteria, and is involved in removal of Okazaki fragments during lagging strand synthesis in E. coli. RNase H is a potential target for anti-retroviral drugs and is an important component of antisense oligonucleotide-based therapeutic approaches. Because protein stability and biological activity are strongly temperature dependent, comparative studies of the temperature dependence of conserved structural and dynamical features of E. coli and T. thermophilus RNase H illuminate the thermodynamic and kinetic principles governing protein structure and function; in particular, E. coli and T. thermophilus RNase H are hypothesized to respond similarly to temperature perturbations at the physical temperature appropriate for each microorganism. To test this and related hypotheses, structures of RNase H-ligand complexes will be determined by multidimensional NMR spectroscopy, changes in conformational dynamics of RNase H in response to temperature changes will be measured by nuclear magnetic spin relaxation and enzyme kinetic parameters for RNase H will be measured as a function of temperature.